Methylprednisolone detection in urine following local and oral administrations
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2 Susana MS Simões, Marta Calçada, Luís Horta, Xavier de la Torre Methylprednisolone detection in urine following local and oral administrations Laboratório de Análises e Dopagem. Instituto do Desporto de Portugal. Av. Prof. Egas Moniz (Estádio Universitário). 6-9 LISBOA. Portugal Introduction Glucocorticosteroids are prohibited when administered orally, rectally, intravenously or intramuscularly. In order to investigate the possibility to distinguish between permitted vs an illicit administration of these substances, we conducted two excretion studies after controlled administrations of methylprednisolone through oral and local administrations. Mehylprednisolone urinary detection as well as the effects on the normal endogenous glucocorticosteroids (cortisol and cortisone) excretion were monitored by routine LC/MS/MS analyses after automatic SPE extraction. Experimental Samples obtained after controlled administrations of methylprednisolone were analyzed following the screening and confirmation procedures for methylprednisolone detection. A brief description of the method used is presented in Figure. Methylprednisolone, methylprednisone, 2α-hydroxymethylprednisolone (see Figure 2), cortisol and cortisone were monitored. Cortisol and cortisone were compared with population based data obtained from routine samples of the Portuguese doping control program. Oral administration: 4 mg of methylprednisolone (Medrol, Pfizer) were given orally to a male volunteer. Local administration: Samples were collected from an athlete (that had a TUE accepted by the National Portuguese Antidoping Council (CNAD). 4 mg of Depo-Medrol, Pfizer by extraarticular infiltation) that volunteered for the study. 4
3 Results After both administrations, concentrations higher than the reporting threshold (3 ng/ml) proposed by WADA are reached (c.a. h for the oral administration and 36 h for the local one) with slightly higher peak concentration after the local administration. Methylprednisolone can be detected up to 32 h after oral administration and 8 h after local administration. The excretion profile of the 3 metabolites monitored is similar. The relative response of 2α-hydroxymethylprednisolone is in both administrations higher than methylprednisolone itself being a more sensitive marker of methylprednisolone intake. After both administrations a deep suppression of cortisol and cortisone concentrations is observed. The lowest concentrations are below the 5 percentile of the population based data (Figures 3-4 and Table ) reflecting the systemic effect reached in both cases. Conclusions After both oral and local administrations, a systemic effect is observed through the inhibition of the endogenous excretion of cortisol and cortisone. The concentrations reached in the permitted administration (dosage s the oral administration) were higher than after an oral administration. The urinary excretion profile of methylprednisolone and its metabolites is similar in both administrations do not permitting to differentiate the route of administration. Figure. Description of the method used. 3 ml of urine Add.3 µg of methyltestosterone (internal standard) ml of sodium acetate buffer.2 M ph 5.2 and 5 µl of β-glucuronidase/ arylsulfatase Incubate the samples at 55ºC for 2 h Condition the Oasis HLB columns with 3 ml of methanol and 3 ml of water Load 4 ml of the samples into the columns Wash the columns with 3 ml of sodium hydroxide.2m: methanol (6:4, v/v) and 3 ml of water Elute the samples with 3 ml of tert-butylmethyl ether: methanol (9:, v/v) Evaporate to dryness under nitrogen at 45 C. Reconstitute in µl of mobile phase Transfer the mixture to a 2 µl vial, and analyse the sample by LC/MS/MS Instrument Waters Alliance 2795 pump/ Micromass Quattro micro TM Column Waters-XTerra MS C8 (5 mm, 2. mm, 5 µm) Flow parameters solvent A ACN/HCOOH. (95:5) solvent B HCOOH./ACN (95:5) flow rate.3 ml/min 65 Mass spectrometric parameters acquisition mode MRM function -6.5 min ( ) cone 2V coll 5eV ( ) cone 2V coll 5eV ( ) cone 2V coll 5eV function min ( ) cone 2V coll 5eV ( ) cone 2V coll 5eV ( ) cone 2V coll 5eV ( ) cone 2V coll ev ( ) cone 2V coll ev ( ) cone 2V coll ev function 2-6 min ( ) cone 35V coll 22eV ( ) cone 35V coll 22eV 42
4 Figure 2. LC/MS/MS analysis of a urine sample collected c.a. 2 h after the infiltration I. 375>357; 375>339 Methylprednisolone II. 373>355; 373>337 Methylprednisone III. 377>28; 377>359-2α-Hydroxymethylprednisolone III I II > e > e > e > e > e > e Time Table. Non-parametric data of population distribution of cortisol and cortisone in routine samples (n=82) 5 Median Mean 95 Cortisol (ng/ml) Cortisone (ng/ml) (5, 95: percentiles) 43
5 Figure 3. Relative urinary detection of methylprednisolone and metabolites after oral and local administrations Oral (4mg) Infiltration (4mg) Metilprednisolona Metilprednisona (met.) 2a-Hidroximetilprednisolona (met.) 6 4 Metilprednisolona Metilprednisona (met.) 2a-Hidroximetilprednisolona (met.) A/A(IS)* 25 2 A/A(IS)* Figure 4. Urinary methylprednisolone and effect on the HPA axis after oral and local administrations Conc. (ng/ml) Methylprednisolone Oral 4 mg (p.o.) Methylprednisolone Hydrocortisone Cortisone Conc. (ng/ml) Methylprednisolone Infiltration 4mg Methylprednisolone Hydrocortisone Cortisone 44
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